Odontophrynus toledoi, Moroti & Pedrozo & Severgnini & Augusto-Alves & Dena & Martins & Nunes & Muscat, 2022

Moroti, Matheus de Toledo, Pedrozo, Mariana, Severgnini, Marcos Rafael, Augusto-Alves, Guilherme, Dena, Simone, Martins, Itamar Alves, Nunes, Ivan & Muscat, Edelcio, 2022, A new species of Odontophrynus (Anura, Odontophrynidae) from the southern portion of the Mantiqueira mountains, European Journal of Taxonomy 847 (1), pp. 160-193 : 166-184

Odontophrynus toledoi sp. nov.

urn:lsid:zoobank.org:act:E7AE4CE9-728E-438A-AF75-8C6EC6C84306

Figs 1–2

Odontophrynus americanus – Savage & Cei 1965. — Beçak et al. 1966. — Araújo et al. 2009. — Lyra et al. 2017.

O. aff. americanus 1 – Martino et al. 2019.

Odontophrynus aff. a mericanus – Rosset et al. 2021.

Diagnosis

Odontophrynus toledoi sp. nov. is a medium-sized species belonging to the genus Odontophrynus based on the phylogenetic position and a combination of morphological characters: granular skin on the dorsum and venter, head wider than long, snout truncate in profile, tympanum hidden, first subarticular tubercle on toe I enlarged, inner metatarsal tubercle large, tarsal fold short ( Savage & Cei 1965; Caramaschi & Napoli 2012). The new species belongs to the Odontophrynus americanus species group based on phylogenetic affinities and the combination of the following characters: absence of large dorsal, tibia and forearm glandular warts, with postorbital, temporal, and parotoid glandular warts not distinctly developed but with a series of small glandular warts of irregular size and shape, forming glandular ridges longitudinally oriented, on postorbital-parotoid regions ( Caramaschi & Napoli 2012). Odontophrynus toledoi is distinguished from the remaining species belonging to the O. americanus group by the following combination of characters: (1) medium sized (SVL = 40.4–51.8 mm in males and 45.0– 54.5 mm in females of O. toledoi ; Table 1 ); (2) head wider than long (HW/HL = 1.31); (3) dorsal surface of head, arms, body and limbs dark brown with arms and limbs with light brown stripes; (4) light mid-dorsal stripe present or interrupted in most of the specimens; (5) yellowish stripe between the eyes, resembling a ‘)’ shape; (6) increased number of longitudinally oriented dorsal glandular ridges; (7) karyotype with 2n = 4X = 44, with fundamental number = 88; (8) advertisement call with dominant frequency of 775–1033 Hz; (9) pulse rate of 89–132 pulses/s; (10) large tadpoles (mean TL = 42.91– 56.18 mm); (11) one–two submarginal papillae on the posterior labium of each side of the oral disc near the posterior emargination; and (12) spiracle sinistral, short, inner wall fused to the body with small distal portion free.

Etymology

The specific epithet honors Professor Luís Felipe Toledo for his contribution in solving the mysteries of the natural history of Neotropical amphibians, especially those from southeastern Brazil and mostly within the Atlantic Forest, where the new species resides.

Type material

Holotype BRAZIL • adult ♂, SVL 49.3 mm; São Paulo State, São José dos Campos, São Francisco Xavier, collected at Rio Manso; 22°54′05″ S, 45°52′43″ W; 1042 m a.s.l.; datum WGS-84; 1 Jan. 2020; E. Muscat and M.T. Moroti leg.; ZUEC-AMP 24833 .

Paratypes BRAZIL – São Paulo State • 1 adult ♀; São José dos Campos, São Francisco Xavier, collected at Projeto Dacnis private reserve; 22°53′45.81″ S, 45°56′30.46″ W; 751 m a.s.l.; datum WGS-84; 15 Jan. 2019; E. Muscat leg.; ZUEC-AMP 24641 • 1 adult ♀; same locality as for holotype; 21 Jul. 2019; E. Muscat and D. Stuginski leg.; ZUEC-AMP 24725 • 2 adult ♀♀; same locality as for holotype; E. Muscat and M.T. Moroti leg.; ZUEC-AMP 24831 • 2 adult ♂♂; same collection data as for preceding; ZUEC-AMP 24834 • 5 adult ♂♂; Santo Antônio do Pinhal; collected at RPPN Fazenda Renópolis; 22°48′20.93″ S, 45°37′32.96″ W; 1332 m a.s.l.; datum WGS-84; 21 Aug. 2019; N.J.S. Fernandes and I.A. Martins leg.; ZUEC-AMP 24763 to 24767 .

Non-type material examined

BRAZIL – São Paulo State • 1 adult ♀; Campos do Jordão, collected at Parque Estadual Campos do Jordão (Horto Florestal); 22°39′55.1″ S, 45°26′59.9″ W, 1531 m a.s.l.; WGS-84; 26 Oct. 2005; I.A. Martins, P.H. Bernardo, F.B.R. Gomes and A.P. Suarez leg.; CCLZU 175 • 2 adult ♂♂; Campos do Jordão, collected at Parque Estadual Campos do Jordão (Horto Florestal); 22°39′55.2″ S, 45°27′12.7″ W; 1520 m a.s.l.; WGS-84; 26 Oct. 2005; I.A. Martins, P.H. Bernardo, F.B.R. Gomes and A.P. Suarez leg.; CCLZU 181-182 • 3 adult ♂♂; Campos do Jordão, collected at Parque Estadual Campos do Jordão (Horto Florestal); 22°41′55.2″ S, 45°27′12.7″ W; 1711 m a.s.l.; WGS-84; 17 Jan. 2005; I.A. Martins, F.B.R. Gomes and A.F. Leite leg.; CCLZU 1484 to 1486 • 1 adult ♀; Campos do Jordão, Parque Estadual de Campos do Jordão (Horto Florestal); 7 Dec. 2009; I.A. Martins, F.B.R. Gomes, C.R. Silva and V.A. Mendes leg.; CCLZU 2637 • 1 adult ♂; same collection data as for preceding; CCLZU 2638 . – Minas Gerais State • 1 adult ♂; Itamonte ; 22°16′58.85″ S, 44°52′11.56″ W; 905 m a.s.l.; WGS-84; 7 Sep. 2004; I.A. Martins leg.; CCLZU 1487 • 1 adult ♀; Cristina, collected at Mata da Prefeitura; 22°13′05″ S, 45°15′25″ W; 1150 m a.s.l.; WGS-84; 25 Aug. 2005; A.F.B Junqueira leg.; CCLZU 2040 • 1 adult ♂; Cristina, collected at Mata da Prefeitura; 25 Aug. 2005; A.F.B Junqueira leg.; CCLZU 2023 • 1 adult ♀; Maria da Fé ; 22º18′15.5″ S, 45°22′43.2″ W; 1265 m a.s.l.; WGS-84; 15 Sep. 2007; A.F.B. Junqueira and F.B.R. Gomes leg.; CCLZU 2525 • 1 adult ♂; Maria da Fé ; A.F.B. Junqueira and F.B.R. Gomes leg.; CCLZU 2526 • 2 adult ♀♀; Marmelópolis ; 22°30′31.9″ S, 45°09′00″ W; 1570 m a.s.l.; datum WGS- 84; 24 Sep. 2009; I.A. Martins leg.; CCLZU 2918, 2919 • 1 adult ♀; Marmelópolis ; 13 Nov. 2009; I.A. Martins leg.; CCLZU 2964 • 1 adult ♂; Marmelópolis ; 22 Jan. 2010; I.A. Martins leg.; CCLZU 3015 • 1 adult ♀; Inconfidentes ; 27 May 2014; Souza J. leg.; ZUEC-AMP23469 .

Description of the holotype

Adult male, body stout, SVL 49.3 mm ( Fig. 1 , Table 1 ). Head wider than long (HW 41.1% of SVL, HL 33.2% of SVL, HW/HL = 1.23). Snout rounded in dorsal view and truncate in lateral view. Canthus rostralis slightly distinct, concave in dorsal view. Loreal region slightly concave. Nostrils elliptic elongated, directed dorsolaterally, and situated at the tip of the snout in lateral view. Internarial distance smaller than eye to nostril distance (IND 71.4% of END) and smaller than interorbital distance (IND 31.2% of IOD). Eyes large, prominent and laterally oriented (ED 32.3% of HL). Upper eyelid with one elongated glandular wart and with a glandular ridge along its marginal edge. Tongue rounded, approximately half free and notched posteriorly. Vomerine teeth in two patches between and the in same line of the choanae. Vocal slits present, longitudinal. Vocal sac well developed, median, subgular. Tympanum hidden, not visible externally. Dorsum of head, body, and limbs with scattered distributed glandular warts, more abundant in flanks. Anterior half of dorsum with series of distinct elongated glandular warts with irregular size and shape, forming small discontinuous pairs of ridges longitudinally oriented behind the postorbital region, parotoid region and in the interscapular region. Skin of venter uniformly covered with small and rounded glandular warts. Forelimbs stout (AL 50% of SVL) covered with small glandular warts, except on dorsal surface of fingers. Ventrolateral surface of forearms with longitudinal series of two elongated glandular warts, fused into ridge. Fingers slender with rounded tips; dermal fringes absent; interdigital webbing absent. Length of fingers: IV>II>V≥III. Subarticular tubercles large, nearly bilobated, proximal more developed than distal. Supernumerary tubercles rounded to oval, covering the palmar surface. Nuptial pads present on thumbs and posterior surface of inner metacarpal tubercles. Inner and outer metacarpal tubercles large, inner oval and outer longitudinally divided, internal part oval, external part elongated. Nuptial pads present on thumbs and posterior surface of inner metacarpal tubercles. Hind limbs stout and relatively short. Tarsal fold present, slightly curved, as long as inner metatarsal tubercle and approximately same length as tarsus. Toes slender with rounded tips; dermal fringes slightly developed; interdigital webbing present. Lengths of toes: IV>III≥V>II>I. Webbing formula: I 1–2 II 1–3 III 2–4 IV 4–2 V. Inner metatarsal tubercle large (IMT 19.3% of FL), shovel-like, with external border keratinized; outer metatarsal tubercle slightly distinct; subarticular tubercles rounded, subarticular tubercle of toe I and II, enlarged, greater than others. Supernumerary tubercles small and rounded, aligned, and covering the plantar surface.

Coloration of the holotype in life

Dorsal surface of head, arms, body, and limbs dark brown ( Fig. 2 ). Arms and limbs with light brown blotches resembling stripes over a dark brown background. A horizontal yellowish stripe between the eyes, resembling a ‘)’ shape. Yellowish dorsolateral stripes, starting behind the head towards the hind limbs. A yellow interrupted mid-dorsal stripe. Irregularly sized light and dark brown blotches distributed below the dorsolateral yellowish stripe. Upper lip yellowish interrupted by dark brown blotches. Vocal sac moss green to black. Venter whitish scattered with grey blotches gradually increasing on the side of the body. Nuptial pads light brown. Foot with outer metatarsal, subarticular, and supernumerary tubercles gray over light brown background. Tarsal fold grayish. Inner metatarsal tubercle light brown with keratinized dark brown portion. Iris with three marbled colors: golden in the dorsal region, black in the middle and whitish in ventral region; pupil horizontally elliptical with golden margins.

Coloration of the holotype in preservative

Dorsal background color predominantly dark brown. Light brown blotches on the arms and limbs. The yellowish stripes became light brown; the whitish or grayish coloration of the antebrachial glandular wart, tubercles of the hand and foot, and tarsal fold became cream-colored. Venter yellowish-colored, with scattered dark brown blotches gradually increasing on the sides of the body. Gular area with dark grey pigmentation.

Variation of adult specimens

Males of O. toledoi sp. nov. differ from females by the presence of a black pigmented gular region on the vocal sac, and well-developed nuptial pads on thumb and posterior surface of inner metacarpal tubercles. Adult females are usually bigger than adult males. Longitudinally oriented glandular warts and the light brown blotches varied in number, size, and shape among individuals. The ‘)’ stripe between the eyes can be poorly distinguished in some individuals and one individual had a distortion, interrupting the stripe. Mid-dorsal stripe is discontinuous and can begin on the head or not.

Tadpoles

Larvae external morphology

Tadpoles of Odontophrynus toledoi sp. nov. have a total length of 42.91–56.18 mm (47.71 ± 5.34 mm) in Gosner stages 37–40 (Lot ZUFMS-AMP 15276, Fig. 3 , Table 2). Body is depressed (BH/BW = 0.85–0.86; Fig. 3A–C ), rounded in lateral view (BL/BH = 1.81–1.86), elliptical in dorsal view (BL/ BW = 1.55–1.58), and slightly longer than one third of the total length (BL/TL = 0.36–0.37). Snout is rounded in lateral view and ovoid in dorsal view. Eyes dorsal, dorsolaterally oriented. Nostrils dorsal with oval shape, anterolaterally positioned with an elevated marginal rim, undeveloped inner margin projection, and closer to the tip of the snout than the eye (NSD/END = 0.77–0.79; Fig. 3G ). Spiracle sinistral, short, inner wall fused to the body with small distal portion free, lateroventrally positioned, posterodorsally directed, and placed at half of the body length (SSD/BL = 0.57–0.60; Fig. 3F ). Ventral tube median, dextral opening, ventrally directed, fused to the ventral fin with posterior portion free and positioned slightly below ventral margin ( Fig. 3D–E ). The dorsal membrane of the ventral tube is slightly shorter than the ventral membrane ( Fig. 3D ). Intestinal mass is circular, located at the center and slightly displaced to the left from the abdomen ( Fig. 3C ). Tail has acute tip, comprising 62.3% of the total length and higher than the body (MTH/BH = 1.02–1.06). Tail musculature is slightly developed (TMH/BH = 0.46–0.51), myotomes visible and more developed on the anterior third and mid portion, and reach the tip of the tail. Dorsal fin has slightly more convex margin than the ventral fin. Dorsal fin high (DFH/TMH = 0.83–1.00) and ventral fin high (VFH/TMH = 0.52–0.60) with maximum height at the mid of the tail. Dorsal fin emerges at the posterior third of body, and the origin of ventral fin is at the inferior margin of ventral tube. Lateral line system has four neuromasts, almost indistinguishable, accumulated near the ventral tube, and others that we could delimit as: supraorbital around the eyes (SO); infraorbital around the snout and nares (IO); posterior supraorbital (PSO); posterior infraorbital near the musculature insertion (PIO); dorsal near the middle of tail musculature (D); and middle (M) on the half of body side ( Fig. 3H– I ).

Oral disc small (ODW/BW = 0.29–0.30) anteroventrally positioned, laterally and posteriorly emarginated ( Fig. 3A, J–K ). Anterior labium has one single row of laterally long marginal papillae aligned on the top and alternated near the emargination with a wide gap anteriorly equivalent to P-2. Papillae aligned, reduced and spaced in lateral emargination. Posterior labium has one single row of long marginal papillae alternated without pigmentation and some bifid aligned papillae, about 14 papillae/mm with four bifid papillae (estimated on the posterior labium; Fig. 3J ). There are one–two submarginal papillae on the posterior labium of each side of the oral disc near the posterior emargination. Submarginal papillae may be absent or not visible during initial stages (e.g., 30–37; Gosner 1960). Labial tooth row formula (LTRF) is 2(2)/3(1) with a short gap in A-2 and P-1; A-1 slightly shorter than A-2; P-1 and P-2 have the same size, both longer than P-3 ( Fig. 3J–K ). Labial teeth dark-colored arranged in a single row, one per tooth ridge with variable size. There are about 52 labial teeth/mm (estimated on P-3). Jaw sheaths serrated, heavily keratinized (with about 37 serrations/mm), and dark with lower sheath base ranging from brown to dark yellow. Upper jaw is arc-shaped with long lateral projection and lower sheath V-shaped, with lower jaw sheath slightly wider than the upper one ( Fig. 3K ).

Coloration of tadpoles in life

Body greenish in life with scattered dark blotches distributed all over it and concentrated on the lateral view of the body. Ventral region cream. Tail light cream with dark blotches, with concentration on last third of the tail. Musculature of the caudal portion easily seen due its reddish coloration ( Fig. 4A ). Mid dorsal cream stripe present in imagoes dorsum ( Fig. 4B ).

Coloration of tadpoles in preservative

Body coloration in preservative 10% formalin is light yellow or cream on the lateral view and dark yellow on dorsal view with melanophores located mainly near the eyes, nares, and on the anterior insertion of tail musculature on dorsal view. There are some angular patches on tail musculature and dorsal and ventral fins on the lateral view. Fins translucent with iridophores, mainly on the dorsal fin. The intestinal region is translucent with small melanophores near to oral disk.

Osteology

Odontophrynus toledoi sp. nov. has a semicircular shaped skull ( Figs 5–6 ), wider than long (SKW/ SKL = 1.41 ± 0.19; Table 3 ) and more elevated posteriorly. Frontoparietal relatively large, broad (width of frontoparietal 5.04 mm ± 0.59 mm), anterior margin rounded, posterior margin crescent-shaped, in contact medially without exposing frontoparietal fontanelle. Nasals triangular, wider than long, keeled, slightly separated from one another medially and well separated from the frontoparietal posteromedially; maxillary process of the nasals in contact with the pars facialis of the maxilla, and with the nasal process of the pterygoids. Maxillary arch complete. Short and wide alary process of the premaxilla. Pars facialis of maxillae bearing around 32 teeth, pars palatina broad, pterygoid process well developed, reaching the level of quadratojugal, wide anterior portion oriented to nasal and posterior portion that contacts maxillary process of nasals. Quadratojugals well developed, contacting maxilla. Vomers moderately sized, separated medially, prechoanal, postchoanal and anterior processes of the vomer well developed, anterior process contacts maxillary arch, dentigerous process of vomer bearing 4–5 teeth. Parasphenoid triradiate, cultriforme process pointed with four peaks, alae perpendicular to cultriform process. Zigomatic ramus of the squamosal moderately sized, anteroventrally directed, separated from the maxilla, enlarged posteriorly and rounded at the anterior end; otic ramus longer than zygomatic ramus. Sphenethmoid well developed, extending anteriorly to the anterior margin of the nasals. Palatines large, strong, narrowly separated medially, expanded laterally. Pterygoides triradiate, relatively large; anterior ramus long connected with maxilla, extending anteriorly to palatine, dorsally curved reaching maxillary process of nasals; medial ramus in contact with otic capsule. Prootic and exoccipital well developed. Occipital condyles large and broadly separated. Crista parotica broad and quadrangular. Posteromedial hyoid process long and well ossified. Vertebral column with 8 presacral vertebrae, I and II imbricate;

transverse process of the presacral II directed anteriorly; transverse processes of presacral II–IV broader and longer than those of presacral V–VIII. Sacral diapophyses moderated dilated. Pectoral girdle arciferal. Clavicle arched directed forward. Ischia well developed. Pubis calcified. Phalangeal formula of hand: 3-3-4-4. Phalangeal formula of foot: 3-3-4-5-4. Pelvic girdle V-shaped.

Vocalization

The advertisement call of Odontophrynus toledoi sp. nov. ( Fig. 7A ) is characterized by a pulse group containing 43‒82 pulses (average = 62 ± 7.3 pulses) occupying a frequency range between 395 and 1399 Hz and dominant frequency range 775 and 1033 (average = 876 ± 83 Hz). Call duration ranges from 438 to 831 ms (average = 607 ± 118 ms). Pulse duration ranges from 4 to 8 ms (average = 5 ± 1 ms) and interval between pulses is 3 ± 0.8 ms (2–5 ms). Pulse repetition rate ranges from 89 to 132 pulses per second (average = 107 ± 13.5 pulse/s) ( Table 4). For complete comparisons between species see Table 5.

Karyotype

Odontophrynus toledoi sp. nov. is a tetraploid species, with 2n = 4X = 44, with all chromosomes biarmed (FN = 88). The karyotype is composed of five metacentric chromosome groups (1, 5‒7, and 11) and six submetacentric chromosome groups (2‒4 and 8‒10; Fig. 8A ). In cells in meiosis I, tetravalents were commonly seen, although in variable numbers ( Fig. 8B ). In Giemsa-stained metaphases, secondary constrictions were observed in the long arm of chromosomes 9, which corresponded to silver-stained NORs revealed by the Ag-NOR method. A notable variation was observed in NOR size in specimen ZUEC-AMP 24764. While three of its chromosomes 9 had large silver-stained NORs, which were easily seen as secondary constrictions in Giemsa-stained metaphases, one chromosome 9 had a small NOR ( Fig.8C ), hardly seen in some metaphases. Such variation in NOR size justifies the differences observed in chromosome 9 length ( Fig. 8A, C ). Constitutive heterochromatin was detected in the centromeric region of all chromosomes ( Fig. 8D ).

Phylogenetic inferences and mitochondrial DNA divergences

Our gene tree for 16S mtDNA recovered the Odontophrynus americanus species group as monophyletic, with low support (0.77 posterior probability) and as a sister clade of the O. occidentalis and O. cultripes species group ( O. cultripes and O. carvalhoi ). Also, our gene tree recovered Odontophrynus toledoi sp. nov. as part of the O. americanus group (0.97 posterior probability). The clade of O. americanus group is composed by three small clades with the following relationship: O. reigi on the basis, as sister taxon of the remaining species, and ( O. cordobae [ O. americanus + O. lavillai ]) as sister clade of ( O. toledoi [ O. juquinha + O. aff. juquinha ]). The two delimitation analyses, bGMYC and BPP, were almost entirely congruent with each other, supporting the existence of nine and ten species, respectively, in the clade of the Odontophrynus spp. ( Fig. 9 ), which mostly agrees with the current taxonomy of the group. Furthermore, the mitochondrial haplotype network shows that O. toledoi does not share haplotypes with other species of the O. americanus species group ( Fig. 10 ).

Geographical distribution

The distribution of Odontophrynus toledoi sp. nov. comprises the southern portion of the Serra da Mantiqueira and adjacent areas that share species with the mountain range, such as Jundiaí and Atibaia in the state of São Paulo. The occurrence area covers two states in Brazil (São Paulo and Minas Gerais; Fig. 10 ). The type locality is São Francisco Xavier (SFX; district of the municipality of São José dos Campos, São Paulo) in the ‘Rio Manso’ area. The species can also be found in the Projeto Dacnis private reserve and in other flooded areas within the district. In the state of São Paulo, the municipalities of Campos do Jordão (Parque Estadual de Campos do Jordão (Horto Florestal), Santo Antônio do Pinhal (RPPN Fazenda Renópolis) São Bento do Sapucaí, Caçapava, Pindamonhangaba, Piquete and Queluz (Martins, I.A. pers. com.) are also in its range. In the state of Minas Gerais, the species can be found in the municipalities of Airuoca, Itamonte, Cristina (Mata da Prefeitura; Fazenda Pouso Frio), Maria da Fé (Fazenda Pomária), Poços de Caldas, Rio Preto, Inconfidentes and Marmelópolis.

Natural history

Odontophrynus toledoi sp. nov. is a terrestrial, nocturnal species commonly found in open and flooded areas. In the type locality (SFX), it was found mainly in valleys. In these locations, ephemeral wetlands receive more water from rivers and precipitation, especially during the rainy season (October–March). Although the frogs are predominantly nocturnal, they can also be heard during daylight on rainy days. They spend most of their time buried in aestivation and generally only come out after heavy rains. We also observed that females leave the aestivation period before males. Females emerge from holes approximately 7cm deep at the beginning of the rains, while males are only observed when there is a greater accumulation of precipitation.

When noticing our presence, the species exhibited the defensive behaviors of burrowing ( Fig. 11A–B ), puffing up the body, stiff leg ( Fig. 11C ). Thanatosis occurred when they were handled. The species’ coloration also provides camouflage in its environment ( Fig. 11D ). It is an explosive breeder and males cluster around temporary pools. Adult males can be seen calling with the body partially submerged in flooded areas, hidden in vegetation. The amplexus is axillary. Tadpoles are exotrophic, benthonic and inhabit mainly lentic environments, such as temporary ponds (ecomorphological guild II: A: 1; sensu Altig & McDiarmid 1999).

Comparison with other species

Odontophrynus toledoi sp. nov. is clearly distinguished from the species of the O. cultripes group ( O. cultripes , O. carvalhoi , and O. monachus ) and O. occidentalis by the absence of distinctly enlarged postorbital, temporal, and parotoid glandular warts (three or more pairs of enlarged rounded-oval postorbital, temporal, and parotoid glandular warts in O. cultripes and O. occidentalis species groups).

Odontophrynus toledoi sp. nov. is distinguished from O. maisuma by its smaller adult females SVL (37.9– 43.6 mm in O. maisuma ; Rosset 2008). From O. americanus , O. cordobae and O. lavillai , O. toledoi can be differentiated by the sloping snout in lateral view (truncate or obtuse in O. americanus [ Martino & Sinsch 2002; present study] and truncate, poor sloping, in O. lavillai [ Rosset et al. 2009]). Odontophrynus toledoi can be distinguished from O. lavillai , O. maisuma and O. reigi by the presence of fringes on fingers (absent in these species; Rosset 2008; Rosset et al. 2021), also from O. maisuma by the presence of fringes on toes (absent in this species; Rosset 2008). The new species can be distinguished from O. cordobae , O. juquinha , O. lavillai and O. maisuma by having longitudinally oriented dorsal glandular ridges (scarce or absent in O. cordobae , O. juquinha and O. lavillai , and scarce or absent, except on postorbital and parotoid regions in O. maisuma ), from O. cordobae by having the rostral glandular wart well developed (poorly developed or absent in O. cordobae ), from O. reigi by the eye-nostril glandular wart poorly developed or absent (well developed in O. reigi ). The skin texture of densely scattered big warts on dorsum can distinguish O. toledoi from O. lavillai (small warts scattered on dorsum; Cei 1980; Rosset 2008). The new species can be distinguished from O. americanus , O. cordobae and O. maisuma by the presence of few distinct postorbital and parotoid glands undifferentiated from other glands (glands form a longitudinal ridge in O. maisuma , rounded and curved postorbital and parotoid glands or ridges on O. americanus and O. cordobae ; Savage & Cei 1965; Martino & Sinsch 2002; Rosset 2008). Odontophrynus toledoi also differs from the others by the temporal gland undistinguishable from other glands (distinctly rounded temporal gland in both O. americanus and O. cordobae , and absent in O. maisuma ; Savage & Cei 1965; Martino & Sinsch 2002; Rosset 2008). The toe length can distinguish O. toledoi from O. maisuma (toe I does not reach the subarticular tubercle of the toe II in O. toledoi , and toe I reaches the subarticular tubercle of the toe II in O. maisuma ; Rosset 2008).

In general, external morphology of tadpoles of O. toledoi sp. nov. are very similar to other members of the O. americanus group, O. cultripes group, and O. occidentalis group. However, in comparison with members of the O. cultripes group ( O. carvalhoi , O. monachus , O. cultripes ), tadpoles of Odontophrynus toledoi differ by their larger size, TL = 42.91–56.18 mm at stages 37–40, against O. monachus (TL = 36.9 mm at stage 36; Menegucci et al. 2016), O. cultripes (TL = 41 mm at stage 37; Savage & Cei 1965; Nascimento et al. 2013), with exception of O. carvalhoi (TL = 58.59 at stage 34; Caramaschi 1979; Santos et al. 2017). By lateral and ventral emarginations on the oral disc (lateral in O. monachus and O. carvalhoi ; absent in O. cultripes ). By 1–2 submarginal papillae on the posterior labium of each side of the oral disc (5–9 grouped laterally in both labia in O. monachus ; 2–3 on upper labium and 3 on lower labium in O. carvalhoi ). Furthermore, differentiated from O. monachus by tip of tail acute (rounded in O. monachus ), ventral tube positioned slightly below ventral margin (at ventral margin in O. monachus ), by body rounded in lateral view (globular in O. monachus ). It can also be diagnosed from O. carvalhoi by having the spiracle’s inner wall with small distal portion free (fused in O. carvalhoi ), LTRF 2(2)/3(1) (varies among 2/3(1) and 2(2)/3(1) in O. carvalhoi ), and elliptical body in dorsal view (ovoid in O. carvalhoi ).

In comparison with tadpoles of the O. americanus group ( O. maisuma , O. juquinha , O. cordobae , O. americanus , O. lavillai ), tadpoles of O. toledoi sp. nov. can be distinguished by their smaller size at same or near stages, TL = 42.91–56.18 mm, stages 37–40, O. maisuma (TL = 47 mm at stages 33–36; Borteiro et al. 2010), O. cordobae (TL = 35.9–50.9 mm at stage 37; Grenat et al. 2009), O. lavillai (TL = 55.8 mm at stage 37; see Lavilla & Scrocchi 1991; Fabrezi & Vera 1997; Nascimento et al. 2013), except by being larger than O. juquinha (TL = 24.3–35.7 mm at stages 30–38; Rocha et al. 2017) and O. americanus (TL = 42 mm, stages 38–39; see Fernández & Fernández 1921; Fabrezi & Vera 1997; Nascimento et al. 2013). By body rounded in lateral view and elliptical in dorsal view (globular and ovoid in O. maisuma , O. juquinha ; ovoid in dorsal view in O. cordobae ; globular in dorsal view in O. lavillai and O. americanus ). By by 1–2 submarginal papillae on the posterior labium (4–8 in both sides of oral disc in O. juquinha ; 1–2 submarginal papillae lower/posterior lip in O. cordobae ). By lateral and ventral emarginations on the oral disc (lateral in O. juquinha , O. maisuma , and lateral and subterminal in O. cordobae ). By nostrils with small projection on marginal rim (absent in O. maisuma , O. cordobae ). Moreover, differentiated from O. juquinha by vent tube with posterior portion free (entirely fused in O. juquinha ). It can also be diagnosed from O. maisuma by having the spiracle’s inner wall with small distal portion free (fused in O. maisuma ), by having one-single row of papillae (varies from 1–2 rows on anterior/posterior labium in O. maisuma ), and by tip of tail acute (rounded in O. maisuma ).

Tadpoles of O. toledoi sp. nov. can be diagnosed from O. occidentalis ( O. occidentalis group) by being smaller ( O. toledoi TL = 42.91–56.18 mm, stages 37–40, O. occidentalis TL = 74 mm at stage 37; Savage & Cei 1965; Cei 1987), by one-single row of marginal papillae (two-rows along oral disc in O. occidentalis according to Nascimento et al. 2013). However, O. barrioi ( González et al. 2014) synonym of the O. occidentalis (see Martino et al. 2019) have one-single row of marginal papillae. Furthermore, O. toledoi can be distinguished from species of the O. occidentalis group by its spiracle’s inner wall with small distal portion free (inner wall absent in O. occidentalis / O. barrioi ); and by its acute tip of tail (rounded in O. barrioi / acute in O. occidentalis ; see Martino et al. 2019). These phenotypic variations may be due to plastic responses to environments and predators ( Van Buskirk et al. 1997; Van Buskirk & McCollum 1999) as mentioned by Nascimento et al. (2013) and Martino et al. (2019). Odontophrynus toledoi can be distinguished from O. maisuma and O. reigi by: (1) phalangeal formula of hand and the foot ( O. toledoi has one additional bone in each phalange, compared to other species); (2) skull with the termination of the cultriform process ending in four cusps (two cusps in both O. maisuma and O. reigi ); (3) frontoparietal fontanelle not exposed (exposed in O. maisuma ); (4) nasals and frontoratietal well separated (slightly separated in O. reigi ).

The first two eigenvectors of the PCA describe 79.5 percent of the total variation present on tadpoles’ morphological measures. PC1 show that tadpole of O. toledoi sp. nov. is not associated with any negative or positive axis, while in PC2 O. toledoi is associate to negative axis. However, PCA show nonoverlapping between tadpoles of the genus Odontophrynus in comparison with tadpole of O. toledoi mainly when compared to O. juquinha (more close-related phylogenetically; Fig. 9 ). Also, shows that close-related species occupied distinct positions, and O. maisuma and O. juquinha are morphologically more distant from the O. toledoi ( Fig. 12 ).

The advertisement call of O. toledoi sp. nov. distinguishes it from O. americanus by the lower interval between pulses ( O. toledoi IBP = 2–5 ms; O. americanus 8–8.6 ms) and the higher number of pulses/ call ( O. toledoi P/C = 43–82; O. americanus 39–41). Moreover, O. toledoi differs from O. cordobae by the longer call duration ( O. toledoi CD = 438–831 ms; O. cordobae 421–475 ms), by the higher number of pulses/call ( O. toledoi P/C = 43–82; O. cordobae 48–53) and by its pulse duration ( O. toledoi PD = 4–8 ms; O. cordobae 3.3–3.7 ms). Odontophryns toledoi – calls in the air temperature range of 18–20°C – is distinguished from O. juquinha and O. lavillai by the higher call duration ( O. toledoi CD = 438–831 ms; O. juquinha 322–610 ms, air temperature 19.2°C; O. lavillai 301.5–583 ms, air temperature range 23– 27°C). Odontophryns toledoi differs from O. maisuma by the lower dominant frequency ( O. toledoi DF = 775–1033 Hz; O. maisuma 1124 –1211 Hz). Furthermore, O. toledoi can be distinguished from O. reigi by the longer call duration ( O. toledoi CD = 438– 831 ms; O. reigi 284.5–688.5 ms).

The first two eigenvectors of the PCA describe 73.8% of the total variation present on the acoustic traits of Odontophrys toledoi sp. nov. and O. juquinha , indicating a separate structuring in the advertisement call of the two species in the acoustic-space ( Fig. 7B ).

Lastly, the new species differs from O. carvalhoi , O. cordobae , O. cultripes , O. juquinha , O. lavillai , O. maisuma , O. occidentalis , and O. reigi by being tetraploid (all these species are diploid, with 2N = 22; Saez & Brum-Zorrilla 1966; Becak & Becak 1974; Salas & Martino 2007; Borteiro et al. 2010; Rocha et al. 2017; Rosset et al. 2021). Additionally, the new species differs from O. cordobae , O. juquinha , O. maisuma and O. reigi by having secondary constriction on the chromosomes of group 9, while in these species this is present at pair 4.

Figures

Fig. 1. Odontophrynus toledoi sp. nov., holotype, ♂ (ZUEC-AMP 24833). A. Dorsal view. B. Ventral view. C. Lateral view of the head. D. Ventral view of the foot. E. Ventral view of the hand. Scale bar = 5 mm.

Fig. 2. Odontophrynus toledoi sp. nov., holotype, ♂ (ZUEC-AMP 24833), in life. A. Lateral view. B. Dorsal view. Scale bar = 5 mm.

Fig. 3. Odontophrynus toledoi sp. nov., external morphology of the tadpole at stage 37–40. A–C. Lateral (A), dorsal (B), and ventral (C) views. D–E. Ventral tube. F. Spiracular aperture. G. Keft nare with an elevated marginal rim. H–I. Body on dorsal view and front view respectively indicating visible series of the lateral line system: supraorbital (SO), infraorbital (IO), posterior supraorbital (PSO), posterior infraorbital (PIO), dorsal (D), and middle (M). J. Oral disc closed. K. Oral disc opened. Scale bars: A–C = 5 mm; D–F = 0.5 mm; G = 0.2 mm; H = 2.5 mm; I = 0.2 mm; J–K = 0.5 mm.

Fig. 4. Odontophrynus toledoi sp. nov. in life. A. Tadpole. B. Metamorph.

Fig. 5. Odontophrynus toledoi sp. nov., holotype, ♂ (ZUEC-AMP 24833), skeleton, generated by microtomography. A. Dorsal view of the individual. B. Ventral view of the individual. C. Lateral view of the head and arms. D. Ventral view of the skull. E. Dorsal view of the skull. F. Ventral view of the hand. G. Ventral view of the foot.

Fig. 6. Odontophrynus toledoi sp. nov. (ZUEC-AMP 23469), skull. A. Dorsal view. B. Ventral view.

Fig. 7. A. Advertisement call of Odontophrynus toledoi sp. nov. B. Acoustic-space of Odontophrynus toledoi sp. nov. and Odontophrynus juquinha Rocha, Sena, Pezzuti, Leite, Svartman, Rosset, Baldo & Garcia, 2017, and the respective grid with the position of each semilandmark. The positions of the semilandmarks were obtained in the 3D spectrogram and summarized in a Principal Coordinate Analysis (PCA). With this, the ordering graphic recovered 73.8% of all the acoustic-space variance in the first two PCA axes, resulting in a non-overlapping of the acoustic-space of the two species.

Fig. 8. Karyotype of Odontophrynus toledoi sp. nov. A. Giemsa-stained karyotype. Note the length variation among chromosomes 9 due to the NOR size variation (see text for details). B. Giemsastained chromosomes extracted from two distinct cells in meiosis I. The number of tetravalents differed between both cells. C. Chromosome group 9 from one metaphase of the specimen ZUEC-AMP 24764 sequentially subjected to Giemsa-staining (G) and the Ag-NOR method (Ag-NOR). Arrows indicate secondary constrictions. Note the NOR size variation among the homologues. D. C-banded karyotype. Scale bars = 10 µm.

Fig. 9. Phylogenetic analysis of partial sequences of the 16S mtDNA gene of 10 species of the genus Odontophrynus Reinhardt & Lütken, 1862. Nodes are labeled with the Bayesian posterior probability. Scale bar (0.4) in million years. The black bars on the side represent the species delimitated by the bGMYC and BPP methods. The colored bars represents the haplotype groups shown and map in Fig. 10.

Fig. 10. Geographical distribution of ten species of Odontophrynus Reinhardt & Lütken, 1862 in South America and the respective haplotype network with the colors of each cluster also indicated on the map. Median-joining haplotype network of specimens from genus Odontophrynus based on partial sequences of the 16S mtDNA gene. Each haplotype is represented by a circle whose area is proportional to its frequency. Traits indicate additional mutational steps for branches with more than one mutation. Different colors indicate species-level units. The black dots are median vectors (hypothesized sequences). Highlighted, the altitude map shows the distribution of the genus in central, south, and southeast of Brazil. Abbreviations: BA = Bahia; ES = Espirito Santo; GO = Goiás; MS = Mato Grosso do Sul; MT = Mato Grosso; PR = Paraná; RJ = Rio de Janeiro; SC = Santa Catarina; SP = São Paulo; TO = Tocantins.

Fig. 11. Odontophrynus toledoi sp. nov. displaying defensive behaviors (A–B) of burrowing and (C) puffing up the body and stiff leg synergistically. D. The coloration of the species also provides crypsis by camouflage in the microhabitat where it lives.

Fig. 12. Principal component analysis (PCA) of the linear measures showing distribution of the species in the reduced space composed by the first two principal components. Yellow circle = O. cultripes group, grey circle = O. americanus group, and blue circle = O. occidentalis group. Tadpole illustrations and pictures were obtained from the original species descriptions: O. juquinha image obtained from Rocha et al. 2017; O. maisuma image, from Borteiro et al. 2010; O. monachus image, from Menegucci et al. 2016; O. carvalhoi image, from Santos et al. 2017; O. americanus, O. cultripes, and O. occidentalis, from Savage & Cei 1965; and O. cordobae image, from Grenat et al. 2009). Linear measures used to perform PCA are in the Supp file 1: Table S2.

Tables

Table 1. Measurements (in mm) of the holotype and 10 paratypes of Odontophrynus toledoi sp. nov. The results are presented as the mean ± standard deviation (sd) and minimum–maximum values (range). Abbreviations of morphometric characters are defined in Material and methods.

Morphometric characters	Holotype	Male (n = 6) Mean ± sd Range		Female (n = 4) Mean ± sd Range
SVL	49.3	45.6 ± 4.1	(40.4–51.8)	51.5 ± 4.5	(45.0–54.5)
HL	16.4	14.4 ± 1.3	(13.0–16.7)	16.3 ± 1.2	(15.0–17.9)
HW	20.3	18.8 ± 1.3	(17.6–20.5)	22.0 ± 0.9	(21.2–23.1)
END	3.5	3.4 ± 0.6	(3.0–4.5)	3.7 ± 0.8	(2.9–4.6)
IND	2.5	2.5 ± 0.3	(2.0–2.9)	3.0 ± 0.4	(2.6–3.5)
ED	5.3	4.8 ± 0.6	(3.9–5.4)	5.5 ± 0.6	(5.0–6.3)
IOD	8.0	6.8 ± 0.6	(5.9–7.8)	8.1 ± 0.8	(7.1–8.9)
AL	25.0	22.6 ± 1.7	(19.9–24.7)	25.0 ± 2.2	(21.8–26.3)
HAL	15.5	13.7 ± 1.0	(12.2–14.7)	15.0 ± 1.6	(12.6–16.3)
F3L	4.8	4.3 ± 0.3	(3.9–4.9)	5.0 ± 0.5	(4.5–5.5)
THL	17.9	17.6 ± 1.4	(15.1–19.1)	19.2 ± 2.4	(16.2–22.0)
TbL	16.7	14.8 ± 1.2	(13.2–16.2)	17.4 ± 1.2	(16.0–18.8)
FL	23.8	21.2 ± 1.3	(19.9–23.5)	24.2 ± 1.9	(22.5–26.8)
T4L	11.8	10.3 ± 0.6	(9.4–11.1)	11.9 ± 0.4	(11.6–12.6)
IMT	4.6	4.1 ± 0.6	(3.5–5.1)	4.9 ± 0.5	(4.4–5.6)

Table 3. Measurements(inmm) and proportionsof theholotypeand five paratype males of Odontophrynus toledoi sp. nov. Values presented as mean ± sd, minimum–maximum values (range). See Material and methods for abbreviation definitions.

Odontophrynus toledoi sp. nov.
MIL	47.16 ± 6.36 (35.81–52.91)
SKW	20.36 ± 1.8 (17.14–22.25)
SKL	14.71 ± 2.65 (9.57–17.02)
WFP	5.04 ± 0.59 (4.67–6.01)
LV2	2.39 ± 0.38 (1.87–2.8)
LSV	2.74 ± 0.69 (1.69–3.64)
SDW	8.87 ± 1.3 (7.79–10.9)
SKW/MIL	0.43 ± 0.03 (0.4–0.48)
SKL/MIL	0.31 ± 0.04 (0.27–0.38)
SKW/SKL	1.41 ± 0.19 (1.24–1.79)
LV2/MIL	0.05 ± 0.01 (0.04–0.06)
LSV/MIL	0.06 ± 0.01 (0.04–0.08)